Modern therapeutic efforts are generally focused on the functions of proteins which contribute to many diseases in animals and man. There have been numerous attempts to modulate the production of such proteins by interfering with the function of biomolecules, such as intracellular RNA, that are involved in the synthesis of these proteins. It is anticipated that protein production will thus be inhibited or abolished, resulting in a beneficial therapeutic effect. The general object of such therapeutic approaches is to interfere with or modulate gene expression events that lead to the formation of undesired proteins.
One such method for the inhibition of specific gene expression is the use of oligonucleotides and oligonucleotide analogs as "antisense" drugs. These oligonucleotide or oligonucleotide analogs are designed to be complementary to a specific, target, messenger RNA (mRNA) or DNA, that encodes for the undesired protein. The oligonucleotide or oligonucleotide analog is expected to hybridize with good affinity and selectivity to its target nucleic acid, such that the normal essential functions of the target nucleic acid are disrupted. Antisense therapeutics hold great promise, as evidenced by the large number of oligonucleotides and oligonucleotide analogs that have been evaluated clinically in recent times. See generally, Ciba Foundation Symposium 209, Oligonucleotides as Therapeutic Agents, John Wiley & Sons, 1997. Further, oligonucleotides and oligonucleotide analogs have shown significant promise in the diagnosis of disease, and have also been used extensively as probes in diagnostic kits and as research reagents.
There is therefore a great need for the large scale production of oligonucleotides and oligonucleotide analogs for commercial application. The predominant synthetic regime currently in use for oligonucleotide synthesis is the phosphoramidite method, which is summarized in FIG. 1. Briefly, oligonucleotides are synthesized on a solid-support via sequential reactions (shown as I-v in FIG. 1) in a predetermined order, typically controlled by a computerized pumping system. For example, synthesis typically begins with a nucleoside linked to a solid-support, typically via a linker molecule attached to the 3'-oxygen of the first nucleosidic synthon (as shown in FIG. 1, compound 1). Deprotection (or "cleavage") of the 5'-hydroxyl group is effected by treatment with deprotecting ("deblocking") solution I, 3% dichloroacetic acid (DCA) in dichloromethane, which removes the 5'-O-(4,4'-dimethoxytriphenylmethyl) hydroxyl protecting group to provide 2, having a free 5'-OH group. Such protecting groups are routinely used in oligonucleotide synthesis to allow selective reaction between two functional groups while protecting all other functionalities present in the reacting molecules.
Deprotection of the nucleoside 5'-O-DMT group as described above causes the release of a DMT cation (shown in FIG. 1), which has a characteristic bright red-orange color. Appearance of the colorful DMT cation facilitates monitoring of the coupling efficiency, and also is used by the computer system as a signal to discontinue the flow of 3% DCA deblocking solution.
Deprotection of triarylmethyl protecting groups such as DMT groups under acidic conditions is reported to be a reversible reaction. Therefore, removal of all of the DMT cation from the solid-support is crucial for the success of the deblocking step. Accordingly, in step ii the support is washed with dry solvent, typically acetonitrile (ACN), which removes traces of acidic solution and any trapped DMT-cation.
In step iii, a nucleoside phosphoramidite (3) is premixed with 1-H tetrazole to produce a very reactive P(III) tetrazolide intermediate (4) that reacts almost immediately with the 5'-OH group of (2) generating (5), which has a phosphite triester internucleosidic linkage. The unreacted excess (4) is then washed from the support with dry ACN.
The unstable P(III) species of (5) is then oxidized to a more stable P(V) internucleosidic linkage, such as a phosphodiester or phosphorothioate, to furnish a dimer or higher order support bound species, such as 7. A capping reaction (represented in FIG. 1 as Step v) is then performed to prevent unreacted 5'-OH groups from further extension. Typically, capping is performed with an acylating reagent. These steps are then repeated iteratively until the desired oligonucleotide is obtained. A more detailed treatment of oligonucleotide synthesis, and further representative synthetic procedures can be found in Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, New York, 1991, which incorporated herein in its entirety.
The preceding synthetic regime creates two large molecular weight waste products--one half mole excess of building block 4 (discarded in the process in ACN with 1-H tetrazole as an activator) and the triarylmethyl protecting group used for 5'-OH protection. The latter amounts to approximately 35% weight of the incoming monomeric phosphoramidite unit 3, and is released as triarylmethyl protecting group cation, typically in dichloromethane. Such triarylmethyl protecting groups are usually tailored molecules with distinct reactivity and stability to specific reaction conditions. Thus, they are expensive and add significantly to the cost of the synthetic process.
One critical challenge in the commercialization of oligonucleotide based therapeutic and diagnostic products is the ability to manufacture and market these products at a reasonable cost with minimal environmental impact. One solution to these problems is to minimize the waste of the nucleoside phosphoramidites used in the chemical syntheses. Thus, attempts have been made to recycle the excess unreacted amidite during synthesis of oligonucleotides. See for example Brill W., Tetrahedron Letts., 1994, 35, 3041; Scremin et al., J. Org. Chem., 1994, 59, 1963. However, these techniques have not been reported to have been successfully applied to the large-scale manufacture of oligonucleotides, as is required for research or commercial purposes.
It can be seen that there exists a need for synthetic methods that address the shortcomings of oligonucleotide synthesis discussed above. To date, there has been no report of the capture and use of protecting group cations generated during the deprotection of protected hydroxyl groups in oligonucleotide synthesis. The present invention is directed to this, as well as other, important ends.